A long term objective of our research effort is to define factors that influence the specificity and efficiency of signal propagation by receptors (R) coupled to heterotrimeric G-proteins (G). OVERALL HYPOTHESIS: Signalling efficiency/specificity is determined in part by accessory proteins found in the receptor's microenvironment which segregate a signaling complex to microdomains of the cell, regulate the efficiency and/or specificity of signal transfer, regulate the basal activity of the system and/or provide alternative modes of signal input to G-protein signaling systems that operate independent of a typical GPCR coupling mechanism. Our goals are to define such accessory proteins, their mechanism of regulation and their dysfunction in various diseases. We identified a group of proteins, Activators of G-protein signaling (AGS) 1-3, that directly regulate the activation state of G-proteins by novel mechanisms. This proposal focuses on AGS3 as a template for understanding the role of such proteins in signal processing. In addition, this proposal will also move toward the identification of novel regulators of Go and Gs. SPECIFIC AIM #1 Define the functional role of the TPR domain of AGS3. SPECIFIC AIM #2 Define the regulation of AGS3 by extracellular stimuli and phosphorylation. SPECIFIC AIM #3 Define the role of AGS3 in G-protein signaling. SPECIFIC AIM #4 Identify and characterize receptor-independent regulators of Go and Gs. AGS3 and related accessory proteins provide unexpected mechanisms for regulation of the G-protein activation cycle and have opened up a new area of research related to the cellular role of G-proteins as signal transducers. As such, these proteins and the concepts advanced with their discovery provide unexpected avenues for therapeutics and understanding disease mechanisms.